A droplet actuator typically includes one or more substrates configured to form a surface or gap for conducting droplet operations. The one or more substrates establish a droplet operations surface or gap for conducting droplet operations and may also include electrodes arrange to conduct the droplet operations. The droplet operations substrate or the gap between the substrates may be coated or filled with a filler fluid that is immiscible with the liquid that forms the droplets.
Droplet actuators are used in a variety of applications, including molecular diagnostic assays, such as enzymatic assays. In one example, lysosomal enzyme tests used in newborn testing assays (NBS) may be performed on a droplet actuator. The NBS assays are fluorescent based tests which measure the release of 4-methylumbelliferone (4-MU) or other umbelliferyl derivatives (e.g., 6-hexadecanoylamido-4-methylumbelliferone; HMU) after enzymatic hydrolysis of the substrates. In the droplet operations environment of a droplet actuator, partitioning of 4-MU (or derivatives) between the aqueous phase (i.e., droplet) and the organic phase (filler fluid) may result in a reduction in the assay signal and potential contamination of neighboring samples. Therefore, there is a need for improved methods for reducing partitioning of 4-MU (or derivatives) in droplet-based bioassays on a droplet actuator.